RESEARCH ARTICLE Open Access Larva of the greater wax moth, Galleria

mellonella, is a suitable alternative host for studying virulence of fish pathogenic Vibrio anguillarum Stuart McMillan1, David Verner-Jeffreys2, Jason Weeks2,3, Brian Austin1 and Andrew P. Desbois1* Background: Microbial diseases cause considerable economic losses in aquaculture and new infection control measures often rely on a better understanding of pathogenicity. However, disease studies performed in fish hosts often require specialist infrastructure (e.g., aquaria), adherence to strict legislation and do not permit high-throughput approaches; these reasons justify the development of alternative hosts. This study aimed to validate the use of larvae of the greater wax moth (Galleria mellonella) to investigate virulence of the important fish pathogen, Vibrio anguillarum. Results: Using 11 wild-type isolates of V. anguillarum, these bacteria killed larvae in a dose-dependent manner and replicated inside the haemolymph, but infected larvae were rescued by antibiotic therapy. Crucially, virulence correlated significantly and positively in larva and Atlantic salmon (Salmo salar) infection models. Challenge studies with mutants knocked out for single virulence determinants confirmed conserved roles in larva and fish infections in some cases (pJM1 plasmid, rtxA), but not all (empA, flaA, flaE). Conclusions: The G. mellonella model is simple, more ethically acceptable than experiments on vertebrates and, crucially, does not necessitate liquid systems, which reduces infrastructure requirements and biohazard risks associated with contaminated water. The G. mellonella model may aid our understanding of microbial pathogens in aquaculture and lead to the timely introduction of new effective remedies for infectious diseases, while adhering to the principles of replacement, reduction and refinement (3Rs) and considerably reducing the number of vertebrates used in such studies